Editing Cloning vector

{{Short description|Small piece of maintainable DNA}}
[[File:PBR322 color.svg|thumb|Schematic representation of the [[pBR322]] plasmid, one of the first plasmids widely used as a cloning vector.]]

A '''cloning vector''' is a small piece of [[DNA]] that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for [[molecular cloning|cloning]] purposes.<ref>{{cite web |url=http://www.theodora.com/genetics/#cloningvector |title=Definition of cloning vector |work=Genome Dictionary |access-date=2012-10-18}}</ref> The cloning vector may be DNA taken from a [[virus]], the [[Cell (biology)|cell]] of a higher organism, or it may be the [[plasmid]] of a bacterium. The [[Vector (molecular biology)|vector]] contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of [[restriction site]]s. The vector and the foreign DNA may be treated with a [[restriction enzyme]] that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by [[Ligation (molecular biology)|molecular ligation]].  After a DNA fragment has been cloned into a cloning vector, it may be further [[subcloned]] into another vector designed for more specific use.

There are many types of cloning vectors, but the most commonly used ones are genetically engineered [[plasmid]]s.  Cloning is generally first performed using ''[[Escherichia coli]]'',  and cloning vectors in ''E. coli'' include plasmids, [[bacteriophage]]s (such as [[Lambda phage|phage λ]]), [[cosmids]], and [[bacterial artificial chromosome]]s (BACs).  Some DNA, however, cannot be stably maintained in ''E. coli'', for example very large DNA fragments, and other organisms such as yeast may be used.  Cloning vectors in yeast include [[yeast artificial chromosome]]s (YACs).

== Features of a cloning vector ==
All commonly used cloning vectors in [[molecular biology]] have key features necessary for their function, such as a suitable cloning site and selectable marker.  Others may have additional features specific to their use.  For reason of ease and convenience, cloning is often performed using ''[[E. coli]]''. Thus, the cloning vectors used often have elements necessary for their propagation and maintenance in ''E. coli'', such as a functional [[origin of replication]] (ori).  The [[ColE1]] origin of replication is found in many plasmids.  Some vectors also include elements that allow them to be maintained in another organism in addition to ''E. coli'', and these vectors are called [[shuttle vector]].

===Cloning site===
All cloning vectors have features that allow a gene to be conveniently inserted into the vector or removed from it. This may be a [[multiple cloning site]] (MCS) or polylinker, which contains many unique [[restriction enzyme|restriction sites]].  The restriction sites in the MCS are first cleaved by restriction enzymes, then a [[Polymerase chain reaction|PCR]]-amplified target gene also digested with the same enzymes is ligated into the vectors using [[DNA ligase]]. The target DNA sequence can be inserted into the vector in a specific direction if so desired.  The restriction sites may be further used for [[sub-cloning]] into another vector if necessary.{{citation needed|date=September 2022}}

Other cloning vectors may use [[topoisomerase]] instead of ligase and cloning may be done more rapidly without the need for restriction digest of the vector or insert.  In this [[TOPO cloning]] method a linearized vector is activated by attaching topoisomerase I to its ends, and this "TOPO-activated" vector may then accept a PCR product by ligating both the 5' ends of the PCR product, releasing the topoisomerase and forming a circular vector in the process.<ref>{{cite web |url=http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/topo/The-Technology-Behind-TOPO-Cloning.html |title=The Technology Behind TOPO® Cloning |work=Invitrogen }}</ref>  Another method of cloning without the use of DNA digest and ligase is by [[Site-specific recombination|DNA recombination]], for example as used in the [[Gateway Technology|Gateway cloning system]].<ref>{{Cite book |chapter=Gateway cloning for protein expression |vauthors=Esposito D, Garvey LA, Chakiath CS |title=High Throughput Protein Expression and Purification |series=Methods in Molecular Biology |year=2009 |volume=498 |pages=[https://archive.org/details/highthroughputpr00shar/page/31 31–54] |doi=10.1007/978-1-59745-196-3_3 |pmid=18988017 |isbn=978-1-58829-879-9 |chapter-url-access=registration |chapter-url=https://archive.org/details/highthroughputpr00shar/page/31 }}</ref><ref>{{cite web |url=http://www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/recombination/gateway/ |title=Cloning Methods - Recombination cloning systems |work=EMBL }}</ref> The gene, once cloned into the cloning vector (called entry clone in this method), may be conveniently introduced into a variety of expression vectors by recombination.<ref>{{cite web |url=http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning/Gateway-Technology.html |title=Gateway® Recombination Cloning Technology |work=Invitrogen}}</ref>

===Selectable marker===
A [[selectable marker]] is carried by the vector to allow the selection of positively [[Transformation (genetics)|transformed]] cells.  [[Antibiotic]] resistance is often used as marker, an example being the [[beta-lactamase]] gene, which confers resistance to  the [[penicillin]] group of [[beta-lactam antibiotics]] like [[ampicillin]].  Some vectors contain two selectable markers, for example the plasmid pACYC177 has both ampicillin and [[kanamycin]] resistance gene.<ref name = "Casali_2003">{{cite book | vauthors = Casali N, Preston A  |title= ''E. coli'' plasmid vectors |series=Methods in Molecular Biology |volume=235 |page=23 |url=https://books.google.com/books?id=r6QC0hTwsrwC&pg=PA23 |isbn=978-1-58829-151-6 |year=2003 }}</ref> Shuttle vector which is designed to be maintained in two different organisms may also require two selectable markers, although some selectable markers such as resistance to [[zeocin]] and [[hygromycin B]] are effective in different cell types.  [[Auxotrophic]] selection markers that allow an auxotrophic organism to grow in [[minimal growth medium]] may also be used; examples of these are ''[[Leucine|LEU2]]'' and ''[[URA3]]'' which are used with their corresponding auxotrophic strains of yeast.<ref>{{cite journal | vauthors = Romanos MA, Scorer CA, Clare JJ | title = Foreign gene expression in yeast: a review | journal = Yeast | volume = 8 | issue = 6 | pages = 423–488 | date = June 1992 | pmid = 1502852 | doi = 10.1002/yea.320080602 | s2cid = 15674832 }}</ref>

Another kind of selectable marker allows for the positive selection of plasmid with cloned gene.  This may involve the use of a gene lethal to the host cells, such as [[barnase]],<ref>{{cite journal | vauthors = Yazynin SA, Deyev SM, Jucovic M, Hartley RW | title = A plasmid vector with positive selection and directional cloning based on a conditionally lethal gene | journal = Gene | volume = 169 | issue = 1 | pages = 131–132 | date = February 1996 | pmid = 8635737 | doi = 10.1016/0378-1119(95)00814-4 | url = https://zenodo.org/record/1258531 }}</ref> [[CcdA/CcdB Type II Toxin-antitoxin system|Ccda]],<ref>{{cite journal | vauthors = Bernard P | title = Positive selection of recombinant DNA by CcdB | journal = BioTechniques | volume = 21 | issue = 2 | pages = 320–323 | date = August 1996 | pmid = 8862819 | doi = 10.2144/96212pf01 | doi-access = free }}</ref> and the [[ParDE type II toxin-antitoxin system|parD/parE]] toxins.<ref>{{cite journal | vauthors = Gabant P, Van Reeth T, Drèze PL, Faelen M, Szpirer C, Szpirer J | title = New positive selection system based on the parD (kis/kid) system of the R1 plasmid | journal = BioTechniques | volume = 28 | issue = 4 | pages = 784–788 | date = April 2000 | pmid = 10769758 }}</ref><ref>{{cite journal | vauthors = Kim HG, Kim HS, Hwang HJ, Chung SK, Lee JM, Chung DK | title = Construction of a pTOC-T vector using GST-ParE toxin for direct cloning and selection of PCR products | journal = Biotechnology Letters | volume = 26 | issue = 21 | pages = 1659–1663 | date = November 2004 | pmid = 15604816 | doi = 10.1007/s10529-004-3518-z | s2cid = 10312859 }}</ref>  This typically works by disrupting or removing the lethal gene during the cloning process, and unsuccessful clones where the lethal gene still remains intact would kill the host cells, therefore only successful clones are selected.

===Reporter gene===
Reporter genes are used in some cloning vectors to facilitate the screening of successful clones by using features of these genes that allow successful clone to be easily identified.  Such features present in cloning vectors may be the [[lac operon|''lacZ''α fragment]] for α complementation in [[blue white screen|blue-white selection]], and/or [[marker gene]] or [[reporter gene]]s in frame with and flanking the [[Multiple cloning site|MCS]] to facilitate the production of [[fusion protein]]s.  Examples of fusion partners that may be used for screening are the [[green fluorescent protein]] (GFP) and [[luciferase]].

===Elements for expression===
{{main |Expression vector}}
A cloning vector need not contain suitable elements for the [[Gene expression|expression]] of a cloned target gene, such as a [[Promoter (biology)|promoter]] and [[ribosomal binding site]] (RBS),  many however do, and may then work as an [[expression vector]].  The target [[DNA]] may be inserted into a site that is under the control of a particular promoter necessary for the expression of the target gene in the chosen host.   Where the promoter is present, the expression of the gene is preferably tightly controlled and [[Enzyme induction and inhibition|inducible]] so that proteins are only produced when required.  Some commonly used promoters are the [[T7 phage|T7]] and [[lac operon|''lac'' promoters]].  The presence of a promoter is necessary when screening techniques such as [[Blue white screen|blue-white selection]] are used.

Cloning vectors without promoter and RBS for the cloned DNA sequence are sometimes used, for example when cloning genes whose products are toxic to ''[[Escherichia coli|E. coli]]'' cells.  Promoter and RBS for the cloned DNA sequence are also unnecessary when first making a [[Genomic library|genomic]] or [[cDNA library]] of clones since the cloned genes are normally subcloned into a more appropriate expression vector if their expression is required.

Some vectors are designed for transcription only with no heterologous protein expressed, for example for ''in vitro'' mRNA production.  These vectors are called transcription vectors.  They may lack the sequences necessary for polyadenylation and termination, therefore may not be used for protein production.

==Types of cloning vectors==
A large number of cloning vectors are available, and choosing the vector may depend upon a number of factors, such as the size of the insert, copy number and cloning method.  Large insert may not be stably maintained in a general cloning vector, especially for those with a high copy number, therefore cloning large fragments may require more specialised cloning vector.<ref name = "Casali_2003" />

[[File:PUC19.svg|thumb|The pUC plasmid has a high copy number, contains a multiple cloning site (polylinker), a gene for ampicillin antibiotic selection, and can be used for blue-white screen.]]

===Plasmid===
{{main|Plasmid vector}}
Plasmids are autonomously replicating circular extra-chromosomal DNA. They are the standard cloning vectors and the ones most commonly used.  Most general plasmids may be used to clone DNA inserts of up to 15 kb in size.  One of the earliest commonly used cloning vectors is the [[pBR322]] plasmid.  Other cloning vectors include the [[pUC19|pUC]] series of plasmids, and a large number of different cloning plasmid vectors are available.  Many plasmids have high copy numbers, for example, [[pUC19]] has a copy number of 500-700 copies per cell,<ref name = "Casali_2003" /> and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent manipulation. However low-copy-number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells.<ref>{{cite web |url= http://www.mfa.od.ua/page23.htm |title= Copy number |work= Genetics Institute, Inc. |access-date= 2013-03-06 |archive-url= https://archive.today/20130419075932/http://www.mfa.od.ua/page23.htm |archive-date= 2013-04-19 |url-status= dead }}</ref>

Some plasmids contain an [[M13 bacteriophage]] origin of replication and may be used to generate single-stranded DNA.  These are called [[phagemid]]s, and examples are the [[pBluescript]] series of cloning vectors.

===Bacteriophage===
The bacteriophages used for cloning are the [[Bacteriophage lambda|λ phage]] and [[M13 phage]].<ref>{{cite journal | vauthors = Chauthaiwale VM, Therwath A, Deshpande VV | title = Bacteriophage lambda as a cloning vector | journal = Microbiological Reviews | volume = 56 | issue = 4 | pages = 577–591 | date = December 1992 | pmid = 1480110 | pmc = 372889 | doi = 10.1128/mr.56.4.577-591.1992 }}</ref>  There is an upper limit on the amount of DNA that can be packed into a phage (a maximum of 53 kb), therefore to allow foreign DNA to be inserted into phage DNA, phage cloning vectors may need to have some non-essential genes deleted, for example the genes for [[lysogeny]] since using phage λ as a cloning vector involves only the lytic cycle.<ref>{{cite book | vauthors = Glick BR, Pasternak JJ |year=2005 |title=Molecular Biotechnology Principles and Applications of Recombinant DNA |edition=3rd |publisher=ASM Press |url= https://books.google.com/books?id=Wz3CtTBe9aUC&pg=PA86 |isbn=9781555816124 }}</ref>  There are two kinds of λ phage vectors - insertion vector and replacement vector.  Insertion vectors contain a unique cleavage site whereby foreign DNA with size of 5–11 kb may be inserted.   In replacement vectors, the cleavage sites flank a region containing genes not essential for the lytic cycle, and this region may be deleted and replaced by the DNA insert in the cloning process, and a larger sized DNA of 8–24 kb may be inserted.<ref name = "Casali_2003" />

There is also a lower size limit for DNA that can be packed into a phage, and vector DNA that is too small cannot be properly packaged into the phage.  This property can be used for selection - vector without insert may be too small, therefore only vectors with insert may be selected for propagation.<ref>{{cite book |title=Gene Cloning and DNA Analysis: An Introduction|author= TA Brown|publisher=Wiley-Blackwell |page=100 |isbn= 978-1444334074 |url= https://books.google.com/books?id=Ju8XeJL9Fc4C&pg=PA100 |date= 2010-04-19}}</ref>

===Cosmid===
[[Cosmids]] are plasmids that incorporate a segment of bacteriophage λ DNA that has the cohesive end site (''cos'') which contains elements required for packaging DNA into λ particles. Under apt origin of replication (ori), it can replicate as a plasmid.  It is normally used to clone large DNA fragments between 28 and 45 Kb.<ref name = "Casali_2003" />

===Bacterial artificial chromosome===
Insert size of up to 350 kb can be cloned in [[bacterial artificial chromosome]] (BAC). BACs are maintained in ''E. coli'' with a copy number of only 1 per cell.<ref name = "Casali_2003" />  BACs are based on [[Fertility factor (bacteria)|F plasmid]], another artificial chromosome called the [[P1-derived artificial chromosome|PAC]] is based on the [[P1 phage]].

===Yeast artificial chromosome===
[[Yeast artificial chromosome]] are used as vectors to clone DNA fragments of more than 1 mega base (1Mb=1000kb) in size. They are useful in cloning larger DNA fragments as required in mapping genomes such as in the [[Human Genome Project]]. It contains a telomeric sequence, an autonomously replicating sequence (features required to replicate linear chromosomes in yeast cells). These vectors also contain suitable restriction sites to clone foreign DNA as well as genes to be used as selectable markers.

===Human artificial chromosome===
[[Human artificial chromosome]] may be potentially useful as a gene transfer vectors for gene delivery into human cells, and a tool for expression studies and determining human chromosome function. It can carry very large DNA fragment (there is no upper limit on size for practical purposes), therefore it does not have the problem of limited cloning capacity of other vectors, and it also avoids possible insertional mutagenesis caused by integration into host chromosomes by viral vector.<ref>{{cite journal | vauthors = Kim JH, Kononenko A, Erliandri I, Kim TA, Nakano M, Iida Y, Barrett JC, Oshimura M, Masumoto H, Earnshaw WC, Larionov V, Kouprina N | display-authors = 6 | title = Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 108 | issue = 50 | pages = 20048–20053 | date = December 2011 | pmid = 22123967 | pmc = 3250132 | doi = 10.1073/pnas.1114483108 | doi-access = free | bibcode = 2011PNAS..10820048K }}</ref><ref>{{cite journal | vauthors = Kouprina N, Earnshaw WC, Masumoto H, Larionov V | title = A new generation of human artificial chromosomes for functional genomics and gene therapy | journal = Cellular and Molecular Life Sciences | volume = 70 | issue = 7 | pages = 1135–1148 | date = April 2013 | pmid = 22907415 | pmc = 3522797 | doi = 10.1007/s00018-012-1113-3 }}</ref>

===Animal and plant viral vectors===
Viruses that infect plant and animal cells have also been manipulated to introduce foreign genes into plant and animal cells. The natural ability of viruses to adsorb to cells, introduce their DNA and replicate have made them ideal vehicles to transfer foreign DNA into eukaryotic cells in culture. A vector based on [[SV40|Simian virus 40]] (SV40) was used in first cloning experiment involving mammalian cells. A number of vectors based on other type of viruses like [[Adenoviridae|Adenoviruses]] and [[Papillomaviridae|Papilloma virus]] have been used to clone genes in mammals. At present, retroviral vectors are popular for cloning genes in mammalian cells. In case of plants like [[Cauliflower mosaic virus]], [[Tobacco mosaic virus]] and [[Geminiviridae|Gemini viruses]] have been used with limited success.
[[File:Blue-white test.jpg|thumb|An LB agar plate showing the result of a blue white screen.   White colonies may contain an insert in the plasmid it carries, while the blue ones are unsuccessful clones.]]

==Screening: example of the blue/white screen==
{{main|Blue white screen}}
Many general purpose vectors such as [[pUC19]] usually include a system for detecting the presence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used is the gene coding for ''E. coli'' [[beta-galactosidase|β-galactosidase]], whose activity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate [[X-gal]] (5-bromo-4-chloro-3-indolyl-beta-d-galactoside) into an insoluble, blue product (5,5'-dibromo-4,4'-dichloro indigo). Cloning a fragment of DNA within the vector-based ''lacZα'' sequence of the β-galactosidase prevents the production of an active enzyme. If X-gal is included in the selective agar plates, transformant colonies are generally blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA.

== See also ==
* [[Vector (molecular biology)]]
* [[Plant transformation vector]]
* [[IMAGE cDNA clones]]
* [[fosmid]]
* [[Golden Gate Cloning]]

== References ==
{{Reflist|2}}

[[Category:Genetics techniques]]
[[Category:Molecular biology]]
[[Category:Cloning]]
[[Category:Plasmids]]